Project Summary/Abstract Telomeres are specialized nucleoprotein structures located at the termini of linear eukaryotic chromosomes that are critical for genome stability. The lengths of telomere reserve often play a major role in dictating the replicative life span of cells. Many human diseases are now known to be caused by aberrations in proteins that regulate telomere synthesis. The two strands of telomeres, known as the G- and C-strand, are synthesized sequentially by telomerase and DNA polymerase ?, respectively. Telomerase mediates the extension of the G-strand through reverse transcription of an integral RNA template component. The newly synthesized G-strand in turn serves as the template for the synthesis of the C-strand by the Pol ? complex. A key regulator of telomere DNA synthesis is the Cdc13-Stn1-Ten1 (CST) complex, a conserved RPA-like complex that binds the telomere G-strand with high affinity and sequence-specificty, and that regulates both telomerase and Pol ?. The goal of this research is to understand the mechanisms of the telomerase, Pol ?, and CST complex with respect to the regulation of telomere G and C-strand synthesis. We will utilize factors derived from several Candida species as models. These versions of the three complexes are particularly amenable to biochemical analyses, allowing us to reconstitute several critical interactions that were difficlt to analyze in other systems. Studies of these biochemically tractable factors have led to a series of new and well-defined hypotheses concerning their mechanisms of action. These hypotheses will be tested through an integrated approach that incorporates biochemical, genetic, and single-molecule FRET techniques. The first aim is to dissect the nucleic acid-binding mechanisms of the Est3-TEN complex (comprised of two critical and conserved domains in the telomerase holoenzyme) and assess their contribution to telomerase activity and processivity in vitro and in vivo. The second aim is to dissect the mechanisms of C-strand synthesis by Pol ?, especially with respect to initiation site selection and primer length regulation. The third aim is to characterize the physical interactions between the CST complex and Pol ?, and define the mechanisms by which CST stimulates telomere C-strand synthesis in vitro and in vivo. The targets of these investigations are conserved between budding yeast and humans. The anticipated outcome is a deeper understanding of mechanisms that regulate telomere DNA synthesis, which should inform the development of telomere-based clinical applications.